Bovine viral diarrhea (BVD) virus is classified in the pestivirus genus and Flaviviridae family. It is closely related to viruses causing border disease in sheep and classical swine fever. Infected cattle exhibit “mucosal disease” which is characterized by elevated temperature, diarrhea, coughing and ulcerations of the alimentary mucosa (Olafson, et al., Cornell Vet. 36:205-213 (1946); Ramsey, et al., North Am. Vet. 34:629-633 (1953)). The BVD virus is capable of crossing the placenta of pregnant cattle and may result in the birth of persistently infected (PI) calves (Malmquist, J. Am. Vet. Med. Assoc. 152:763-768 (1968); Ross, et al., J. Am. Vet Med. Assoc. 188:618-619 (1986)). These calves are immunotolerant to the virus and persistently viremic for the rest of their lives. They provide a source for outbreaks of mucosal disease (Liess, et al., Dtsch. Tieraerztl. Wschr. 81:481-487 (1974)) and are highly predisposed to infection with microorganisms causing diseases such as pneumonia or enteric disease (Barber, et al., Vet. Rec. 117:459-464 (1985)).
According to virus growth in cultured cells, two viral biotypes have been classified: viruses that induce a cytopathic effect (cp) and viruses that do not induce a cytopathic effect (ncp) in infected cells (Lee et al., Am. J. Vet. Res. 18: 952-953; Gillespie et al., Cornell Vet. 50: 73-79, 1960). Cp variants can arise from the PI animals preinfected with ncp viruses (Howard et al., Vet. Microbiol. 13: 361-369, 1987; Corapi et al., J. Virol. 62: 2823-2827, 1988). Based on the genetic diversity of the 5′ non-translated-region (NTR) and the antigenic differences in the virion surface glycoprotein E2 of BVD viruses, two major genotypes have been proposed: type I and II. BVDV type 1 represents classical or traditional virus strains which usually produce only mild diarrhoea in immunocompetent animals, whereas BVDV type 2 are emerging viruses with high virulence which can produce thrombocytopenia, hemorrhages and acute fatal disease (Corapi et al., J. Virol. 63: 3934-3943; Bolin et al., Am. J. Vet Res. 53: 2157-2163; Pellerin et al., Virology 203: 260-268, 1994; Ridpath et al., Virology 205: 66-74, 1994; Carman et al., J. Vet. Diagn. Invest. 10: 27-35, 1998). Type I and II viruses have distinct antigenicity determined by a panel of MAbs and by cross-neutralization using virus-specific antisera raised in animals (Corapi et al., Am. J. Vet. Res. 51: 1388-1394, 1990). Viruses of either genotype may exist as one of the two biotypes, cp or ncp virus.
The RNA genome of BVDV is approximately 12.5 kb in length and contains a single open reading frame located between the 5′ and 3′ NTRs (Collett et al., Virology 165: 191-199). A polyprotein of approximately 438 kD is translated from the open reading frame and is processed into viral structural and nonstructural proteins via cellular and viral protease (Wiskerchen et al., Virology 184: 341-350, 1991; Ruemenapf et al., J. Virol 67: 3288-3294, 1993; Elbers et al., J. Virol. 70: 4131-4135, 1996; Tautz et al., J. Virol 71: 5415-5422, 1997; Xu et al., J. Virol 71: 5312-5322, 1997).
The first viral protein encoded by the open reading frame is a protease Npro which cleaves its self from the rest of the polyprotein (Wiskerchen et al., J. Virol 65: 4508-4514, 1991; Stark et al., J. Virol. 67: 7088-7095, 1993). The second protein C is the structural core protein, which packages the genomic RNA and forms the viral virion (Thiel et al., J. Virol. 67: 3288-3294, 1993). Following the protein C-coding sequence are three sequences coding for envelope proteins E0, E1 and E2. E0, E1 and E2 are all glycoproteins. E2 is very antigenic and elicits the production of neutralizing antibodies in the host after infection or vaccination with live or killed vaccines.
A small peptide p7 is located between E2 and the nonstructural proteins. Following p7 is the p125 or NS23 region. NS2 is highly hydrophobic and has a zinc finger motif. NS3 is hydrophilic and is a marker of cytopathic BVDV. NS3 is the most conserved protein in the genus pestivirus and highly immunogenic in infected cells. Replication of a ncp virus in infected animal can convert the virus into the cp biotype through genetic recombination event by insertion of an extra viral or cellular RNA sequence between NS2 and NS3 coding region. As a consequence of the recombination, p125 is processed and free NS2 and NS3 proteins are released (Meyers et al., Nature 341: 491, 1989; Virology 180: 602-616, 1991; Virology 191: 368-386, 1992; Tautz et al., J. Virol. 68: 3289-3297, 1994). NS3 is a viral protease responsible for most of the nonstructural protein processing (Wiskerchen et al., Virology 184: 341-350, 1991). It is also proposed that NS3 plays an essential role in viral RNA replication because of its RNA-stimulated NTPase activity and RNA helicase activity (Tamura et al., Virology 193: 1-10, 1993; Warrener et al., J. Virol. 69: 1720-1726, 1995; Grassmann et al., J. Virol. 73: 9196-9205, 1999). NS4A is located next to NS3 and is known as a cofactor for NS3 protease activity (Xu et al., J. Virol. 71: 5312-5322, 1997). Following NS4A are two viral proteins NS4B and NS5A with unknown functions. The last protein from the open-reading frame of the virus is NS5B, which is a RNA-dependent RNA polymerase and is responsible for viral RNA replication (Young et al., Ogram et al., Fifth International Symposium on Positive Strand RNA Viruses P2-15, P2-16, 1998).
Studies from BVD virus infected animals suggest that BVD viruses induce both B-cell and T-cell responses in animals (Donis et al., Virology 158: 168-173, 1987; Larsson et al., Vet. Microbiol. 31: 317-325, 1992; Howard et al., Vet. Immunol. Immunopathol. 32: 303-314, 1992; Lambot et al., J. Gen. Virol. 78: 1041-1047, 1997; Beer et al., Vet. Microbiology. 58: 9-22, 1997). Both antibodies (Bolin et al., Am. J. Vet. Res. 51: 703-707, 1990).
A number of BVDV vaccines have been developed using chemically inactivated BVD viral isolates (Fernelius et al., Am. J. Vet. Res. 33: 1421-1431, 1972; Kolar et al., Am. J. Vet. Res. 33: 1415-1420, 1972; McClurkin et al., Arch. Virol. 58: 119, 1978). Multiple doses are required for the inactivated viral vaccines to achieve primary immunization. Some inactivated BVDV vaccines provide protection against infection by type I BVDV only (Beer et al., Vet. Microbiology. 77:195-208, 2000). Fetal protection has not been achieved with inactivated BVDV vaccines due to a short duration of immunity and an inefficient cross-type protection (Bolin, Vet. Clin. North Am. Food Anim. Pract. 11: 615-625, 1995).
Modified-live virus (MLV) vaccine, on the other hand, offers a higher level of protection. Currently, licensed BVDV mlv vaccines are produced using attenuated viruses obtained via repeated passage in bovine or porcine cells (Coggins et al., Cornell Vet 51: 539, 1961; Phillips et al., Am. J. Vet. Res. 36: 135-, 1975), or using chemically modified viruses which exhibit temperature-sensitive phenotype (Lobmann et al., Am. J. Vet. Res. 45: 2498-, 1984; 47: 557-561, 1986). A single dose of MLV vaccine is sufficient for immunization, and duration of the immunity can last for years in vaccinated cattle. However, as these vaccines have been developed using type I BVDV virus strains, the full protection is achieved only for type I virus.
There is a need for development of BVDV vaccines that provide protection against both type I and type II viruses. Currently, there are ncp-BVD type II viruses which are candidates for use as an inactivated vaccine based on type II virus isolates (Flores et al., Vet. Microbiology, 77: 175-183, 2000).
The present invention provides genetically engineered type I/type II hybrid viruses using recombinant DNA technology. The present invention further provides immunogenic compositions and vaccines formulated using the genetically engineered hybrid viruses.